12.3 Flashcards
what are reasons for cloning DNA in living cells?
amplification (before PCR)
make more DNA with high fidelity
produce substances form genes
modify genomes of plants or animals
what does cloning require?
a vector - like a plasmid
what do plasmids contain?
origin of replication
selectable markers to identify cells
unique restriction enzyme cleavafe sites
what is a plasmid?
a stable, self-replicating molecule of circular DNA
what is pUC 19?
a typical and common bacterial vector that contains portion of lacZ+ gene with restriction site linker with numerous unique restriction enzyme cut sites and contains ampR (ampicilin resistance gene)
how are foreign DNA sequences inserted into plasmids?
cut foreign DNA with restriction enzyme
cut plasmid with same restriction enzyme
mix cut foreign DNA and cut plasmid DNA
use DNA ligase to seal sugar-phosphate bonds
what are competent bacteria?
E. coli made receptive to transformation by chemical or electrical treatment
and LacZ-
how do you transform competent cells?
using ligated plasmids with DNA inserts
what happens to transformed bacteria?
plated out on agar media with ampicillin and X-gal
what happens do bacteria with no plasmid?
they do not grow because they have no antibiotic resistance
what happens to bacteria with non-recombinant plasmids?
produce b-galactosidase resulting in blue colonies
what happens to bacteria with recombinant plasmids?
do not produce b-galactosidase, resulting in white colonies
what works well with bacterial expression vectors making gene products?
not good with anything that requires post-translational modification
esp. good with enzymes that originate from bacteria
